Goal for the module Lab module 6a Receptor-mediated endocytosis To follow cell surface receptors as they are internalized. Pre-lab homework Read about receptor-mediated endocytosis and other forms of internalization in Alberts Molecular Biology of the Cell, available free online from the National Institutes of Health (http://www.ncbi.nlm.nih.gov/books/bv.fcgi?rid=mboc4.section.2383). Introduction In this module you will use a fluorescent analog of low density lipoprotein (LDL) that binds with high affinity to its cell surface receptor (the LDL receptor or LDL-R). This receptor and it bound ligand is brought into the cell via cell surface clathrin-coated invaginations (coated pits), clathrin-coated vesicles and acidified transport vesicles (endosomes). The receptor and ligand dissociate and the ligand is further transported to lysosomes for degradation. An LDL particle is made up of a single copy of a large receptor-binding protein, apolipoprotein B (apob), a monolayer of phospholipids and cholesterol, and an inner core of cholesterol molecules esterified to fatty acid chains. See Fig. 6.1. LDL is thus rich in cholesterol, making it the bad cholesterol in blood plasma. Lipoprotein particles with much less cholesterol have relatively more protein than lipid, making them higher density. This is the HDL (or good cholesterol) of blood plasma. Fig. 6.1. Each spherical LDL particle has a molar mass of approximately 3 10 6 g/mol (or 3 MDa). It contains a core of about 1500 cholesterol 6.1
molecules esterified to long-chain fatty acids that is surrounded by a lipid monolayer composed of about 800 phospholipid and 500 unesterified cholesterol molecules. A single molecule of a 500,000 Da protein (ApoB) organizes the particle and mediates the specific binding of LDL to its cellsurface receptor protein. (Taken from Molecular Biology of the Cell, 4 th Ed., Alberts et al.) LDL in the blood plasma binds to a specific cell surface receptor found on most cells. The LDL receptor, which is a transmembrane protein, has its LDL binding site on the extracellular side of the cell plasma membrane. One LDL binds one LDL-R. The intracellular side of the LDL-R has interaction sites for an adaptin protein that mediates the binding of the LDL-R/adaptin complex to the protein clathrin that forms a coat on the inner surface of cell surface invaginations called coated pits (see Fig. 6.2). Fig. 6.2. The LDL receptor is a single-pass transmembrane glycoprotein composed of about 840 amino acids (for the human receptor), only 50 of which are on the cytoplasmic side of the membrane. The LDL-R, once inserted into the plasma membrane, diffuses to coated pits where it is captured by the adaptin bound to clathrin. LDL can bind to LDL-Rs in coated pits as well as LDL-Rs that have not yet bound to coated pits. (Taken from Molecular Biology of the Cell, 4 th Ed., Alberts et al.) Coated pits continuously internalize membrane and membrane proteins that are contained within the pits. Thus, LDL-Rs and any LDL that is bound to the receptors are brought into the cell. The coated pits pinch off from the plasma membrane to form coated vesicles, the interior (or lumen) of which contains material that was on the extracellular face of the membrane. Therefore, the internalized LDL is inside these vesicles (see Fig. 6.3). As they are transported inwardly, the clathrin coat is stripped from the vesicles and a proton pump in the vesicle begins to acidify the lumen. The stripped vesicles fuse with other vesicles to form endosomes, which further acidify. The lowered ph in the endosomes causes the LDL to dissociate from the LDL-R. A sorting endosome segregates the freed LDL and the LDL-R. The LDL is then transported to the digestive organelle (the lysosomes) while the LDL-Rs are recycled via recycling endosomes back to the cell surface to repeat their endocytosis cycle. LDL in the lysosomes is degraded to free lipids and amino acids. 6.2
Fig. 6.3. The receptor-mediated endocytosis of LDL follows what is called the degradative or lysosomal pathway. Note that the LDL dissociates from its receptor in the acidic environment of the endosome. After a number of steps the LDL ends up in lysosomes, where it is degraded to release free cholesterol. In contrast, the LDL receptor proteins are returned to the plasma membrane via transport vesicles that bud off from the tubular region of the early endosome, as shown. For simplicity, only one LDL receptor is shown entering the cell and returning to the plasma membrane. Whether it is occupied or not, an LDL receptor typically makes one round trip into the cell and back to the plasma membrane every 10 minutes, making a total of several hundred trips in its 20-hour life-span. (Taken from Molecular Biology of the Cell, 4 th Ed., Alberts et al.) A highly fluorescent analog of LDL will be used in this lab. This analog is identical to the LDL of Fig. 6.1 except that about 50 of the phospholipids in the outer membrane of the LDL particle have been replaced by a fluorescent lipid analog, dii (1,1'-didodecyl- 3,3,3',3'-tetramethylindocarbocyanine perchlorate). You can find the spectral properties of the dii-ldl from the Molecular Probes website. Because there are ~50 fluorophores per LDL particle, it is possible to visualize (even with your eye) individual dii-ldl particles. In this module you will explore the temperature and time dependence of LDL trafficking. In subsequent modules you will compare LDL internalization to the internalization of a different ligand, transferrin, that follows a different internalization pathway and you will neutralize the acidic ph of the endosomal vesicles and monitor the effect that this has 6.3
on internalization. You will also follow the dynamics of LDL vesicle traffic as it moves along microtubules. Materials 3T3 mouse fibroblasts on cover slips LLC-PK pig epithelial cells on cover slips dii-ldl at 10 μg/ml in PBS plus 1 mg/ml BSA (ice temperature) Fine forceps Small beakers Humid chamber(s) Glass slides Fixative solution (PBS plus 3.7% paraformaldehyde) Mounting medium Ice bucket Procedures The dii-ldl reagent that you will be working with is produced from human blood serum. It has been tested and found negative for viruses. Nevertheless, you should wear gloves while working with the labeling solution. Rinse solutions should be disposed of in a bottle that we will autoclave. Anything that may have contacted cells or solutions exposed to dii-ldl (Petri dishes, gloves, Parafilm, tips, filter paper, etc. should be placed in a biohazard bag for autoclaving). Used glass slides can be disposed of in the glass trash. They have been sterilized by the formaldehyde fixation. We do all of this just to be on the safe side. The dii-ldl reagent is expensive. Each lab group will do a different experiment on three separate cover slips, and the data will be shared among groups. We will follow endocytosis of LDL for different times at different temperatures. In addition, we will look at two different cell types, 3T3 cells and LLC-PK cells. Groups 1, 2 and 3 will use 3T3 fibroblasts. Groups 4, 5 and 6 will use LLC-PK cells. ALL GROUPS WILL START WITH THE SAME INITIAL LABELING STEP. AFTER THAT, THE PROTOCOLS DIFFER. Groups 1 and 4 will incubate at 4 C for 1 and 20 min. Groups 2 and 5 will incubate at 25 C (room temperature) for 1 and 20 min. Groups 3 and 6 will incubate at 37 C for 1 and 20 min. In addition, each group will incubate one cover slip at 37 C for different times. A schematic of the time line for these protocols is given below. The essential difference between protocols is the temperature at which incubation happens for 1 and 20 minutes after initial LDL binding to the cell surface. One experiment also looks at different time points of internalization at 37 C, a temperature at which endocytosis is efficient. The purpose of the different incubation times is to follow the time course of internalization of 6.4
the dii-ldl. The purpose of different temperatures of incubation is to examine the role that temperature plays in the endocytosis process. 0 min 20 min 21 min 30 min 40 min 50 min All: label at 0 C 25 min All: incubate at your temp. for 1 and 20 min Grps 1 & 4: 5 min at 37 C Grps 3 & 6: 10 min at 37 C Grps 2 & 5: 30 min at 37 C All groups: initial dii-ldl binding Make three humid chambers for labeling and subsequent incubation. Use ice-cold buffer and place the chambers on ice in an ice bucket. This will prechill the chamber surfaces. Label each chamber with your initials or other identifying mark. Each dish will be incubated in a different way. Label one dish each for each of the three upcoming protocols (see below for your group). You might chose to label these as A, B and C. Place one 50 μl drop of the dii-ldl labeling solution on each humid chamber Parafilm sheet. Only after you have this set up should you obtain 3 cover slips of cells (see above for the cell type for your group). You will use the Petri dishes that the cells are in for subsequent steps. Label each dish as you did with the three humid chambers. One cover slip at a time, remove the growth medium from a cell Petri dish and rinse 3 times with ice-cold buffer. Leave the last buffer rinse in the Petri dish and put it into the ice bucket to keep the cells cold. This will prechill the cells to 4 C. When you have all three cover slips rinsed and chilled, quickly transfer them to the humid chambers to begin the binding of dii-ldl to the LDL receptors on the cell surface. Blot the excess buffer from the cover slips before placing them on the dii-ldl drop. It is important to keep the cells cold. Do not delay in the transfer of the rinsed cells to the humid chamber. Proceed to the protocol for you group. 6.5
Groups 1 and 4: low temperature protocol After the initial 20 minute incubation is complete, you will perform timed incubations of the three cover slips at two different temperatures. This step will allow the dii-ldl to internalize. Leave one Petri dish with cover slip in the ice bath at 4 C for one more minute. Leave one Petri dish with cover slip in the ice bath at 4 C for a 20 minute incubation. Place one Petri dish with cover slip on the 37 C slide warmer for a 5 minute incubation. After each incubation, immediately rinse each cover slip in its appropriate original Petri dish with three changes of ice-cold buffer to stop further processing of the dii-ldl. To do this, place 1 ml of ice-cold buffer in the original Petri dish and quickly transfer the incubating cells to the original Petri dish. (The cell side of the cover slip goes up in the wash Petri dish.) Remove and replace this buffer two times, leaving the last buffer rinse in the Petri dish. Return the dish to the ice bucket. Keep the cells on ice until you can fix the cells in formaldehyde. Once all three cover slips are rinsed, proceed to fixation (below). Groups 2 and 5: room temperature protocol After the initial 20 minute incubation is complete, you will perform timed incubations of the three cover slips at two different temperatures. This step will allow the dii-ldl to internalize. Move one Petri dish with cover slip from the ice bath to a safe location at room temperature for a one minute incubation. Move one Petri dish with cover slip from the ice bath to a safe location at room temperature for a 20 minute incubation. Place one Petri dish with cover slip on the 37 C slide warmer for a 10 minute incubation. After each incubation, immediately rinse each cover slip in its appropriate original Petri dish with three changes of ice-cold buffer to stop further processing of the dii-ldl. To do this, place 1 ml of ice-cold buffer in the original Petri dish and quickly transfer the incubating cells to the original Petri dish. (The cell side of the cover slip goes up in the wash Petri dish.) Remove and replace this buffer two times, leaving the last buffer rinse in the Petri dish. Return the dish to the ice bucket. Keep the cells on ice until you can fix the cells in formaldehyde. Once all three cover slips are rinsed, proceed to fixation (below). 6.6
Groups 3 and 6: 37 C incubation protocol After the initial 20 minute incubation is complete, you will perform timed incubations of the three cover slips at two different temperatures. This step will allow the dii-ldl to internalize. Move one Petri dish with cover slip from the ice bath to the 37 C slide warmer for a one minute incubation. Move one Petri dish with cover slip from the ice bath to the 37 C slide warmer for a 20 minute incubation. Move one Petri dish with cover slip from the ice bath to the 37 C slide warmer for a 30 minute incubation. After each incubation, immediately rinse each cover slip in its appropriate original Petri dish with three changes of ice-cold buffer to stop further processing of the dii-ldl. To do this, place 1 ml of ice-cold buffer in the original Petri dish and quickly transfer the incubating cells to the original Petri dish. (The cell side of the cover slip goes up in the wash Petri dish.) Remove and replace this buffer two times, leaving the last buffer rinse in the Petri dish. Return the dish to the ice bucket. Keep the cells on ice until you can fix the cells in formaldehyde. Once all three cover slips are rinsed, proceed to fixation (below). All groups: fixation Keep the Petri dishes on ice until you can fix the cells in formaldehyde. Do this in the fume hood wearing gloves. The hood is downstairs in room 107. (One group at a time in the hood we will bring you there and show you what to do.) Remove the buffer and replace with 1 ml of the fixative. Fix for 15 minutes. As the cells are now dead, you may mount them at room temperature on clean labeled slides: place a small drop of mounting media on the slide, and then invert the cover slip onto the drop. Blot excess PBS from the cover slip prior to mounting it in Vectashield mounting medium. Seal with nail polish. Now proceed on to the imaging section below. All groups: imaging The goal of this Module is to explore the time and temperature dependence of the endocytosis of ligands bound to their cell surface receptors. Your cells will complete one piece of the whole picture of this process. In order to have a complete picture of the endocytosis of LDL in each cell type, you need to take a set of data that will be compatible with the data from other groups. We want to know several things about the LDL endocytosis process. Among them are 1. The time it takes to deliver LDL from the cell surface to endosomes 6.7
2. The time it takes to deliver LDL from the cell surface to lysosomes. 3. The temperature dependence of these two processes. 4. The distribution of LDL-Rs on the cell surface. 5. The distribution of LDL in endosomes and lysosomes. To get at these points you will observe the distribution of dii-ldl on the surfaces and in the cytoplasm of your cells. You will take through-focal image series to observe the distribution of dii-ldl throughout the cell. You will measure the total amount of dii-ldl in patches on the cell surface and in endosomes and lysosomes. Observe your cells. For each cover slip of cells, observe by eye and using the camera where dii-ldl is detected. DiI has an excitation peak around 540 nm and an emission peak around 580 nm. The green excitation/red emission fluorescence filter cube will work well for dii-ldl imaging. You should get a feel for the distribution of dii-ldl in the cells and the variation among cells before taking and storing images. Endosomes will be in the cytoplasm of the cells. Lysosomes are much larger (i.e. brighter) than endosomes and are often found near the cell nucleus. Try to minimize dii photobleaching by shutting off fluorescence excitation between your observations and also use the shutter when taking single pictures with the camera. Through-focal series. You should take a series of images from the bottom part of the cell to the top of the cell. The 100X, 1.3 NA lens is best for this purpose. The throughfocal imaging can be done in a semi-quantitative way by using the fine focus knob markings. One full rotation of the fine focus control corresponds to 100 μm of movement in the z-direction. Thus, each mark on the fine focus knob equals 1 μm. Find a group of cells that you think is representative of the cell population. Locate the bottom of the cells by focusing on it. While one partner observes the cells, the other partner should change the focus using the fine focus control. Count the number of fine focus marks are needed to scan all the way to the top of the cells. This is the thickness of the cells in μm. (Not all cells will be the same thickness. You should scan to the top of the thickest one in your field of view.) You need to keep the exposure of each of the images in the through-focal series the same as all the rest for subsequent image analysis. You don t want to overexpose very bright elements of any image, so find the brightest area in your image stack and set the fluorescence illumination and shutter exposure time to keep the brightest pixels below the maximum camera signal value (4095). Probably keeping the camera signal below 3500 is a safe bet in case you missed some other very bright feature in one of your slices. Now take a through-focal series of images at 1 μm z-steps from the bottom to the top of the cells. You can do this by taking each image manually as you increment the fine focus knob or you can do this by setting up a time lapse image series (with enough images to cover the number of focal steps that you will need). Start the first image at the bottom of the cells and then move the fine focus in 1 μm intervals between images. Having 10 sec between images for the time-lapse approach should give you enough 6.8
time to change focus after one image has been taken and before the next one is taken. Be sure to use the shutter under computer control for pulsed fluorescence illumination to minimize photobleaching of the dii-ldl when you take these images. Take a phase-contrast image at one of the focal planes to have a reference image for this fluorescence image series. Repeat this for all three sets of cover slips. You might need to reset the image exposure time between cover slips. Make note of these exposure times: they will let you quantitatively compare the different sets of images. Note that you will be taking more pictures of each cover slip in the next step, and you might want to do that now before moving on to the next cover slip. Cell surface and cytoplasmic distribution of dii-ldl. One thing we want to quantitate is the relative number of dii-ldl particles in clusters on the cell surface, how many cell surface clusters there are, the number of endosomes and lysosomes and the brightness of these organelles. As with the through-focal series, set the illumination and exposure time to keep the camera from saturating at any image slice that you might use. Leave these settings fixed for all of the images. Focus on the top of a cell or group of cells (this may be more difficult for the LLC-PK cells). Take an image of the dii-ldl particles bound to LDL-Rs. Now focus in the cell interior and take an image of dii-ldl in endosomes and lysosomes. Use your judgment to select a good focal plane (or planes if you want to take more than one). Repeat this until you have at least 50 individual dii- LDL clusters on cell surfaces and 50 in the cell interiors. You will quantitate the dii-ldl content of these clusters later. You should take companion phase contrast pictures for each field of cells. Repeat this for all three sets of cover slips. It will be best if you can leave the camera exposure settings constant for all three cover slips, but adjust them if you run into a camera overexposure situation. Be sure to keep track of the exposure time to allow quantitative comparisons of dii-ldl content in subsequent image analysis. Analysis Through-focal series: Make a single projection image of each through-focal stack. Read in the through-focal images into a single image stack (File>Import>Image sequence ). Set the pixel-to-distance conversion in the images with the Set scale menu item. Now compress the image stack into a single projection image (Image>Stacks>Z project ) using one of the Projection types. Average Intensity stores average intensity of all images in the stack at each pixel location. Maximum Intensity projection creates an output image each of whose pixels contains the maximum value over all images in the stack at the particular pixel location. Minimum Intensity projection creates an output image each of whose pixels contains the minimum value over all images in the stack at the particular pixel location. Sum Slices creates an image that is the sum of the slices in the stack. Standard Deviation creates an image containing the standard deviation of the slices. Median stores the median 6.9
intensity of all images in the stack at each pixel location. Your best bet for the bright dots of dii-ldl is probably the Maximum Intensity projection, though you should try other projections to see what they produce. This sort of image processing essentially extends the focal distance of the microscope by artificially keeping the brightest elements of each image and throwing away the rest. Most of the brightest elements will be those that are in best focus in each slice. Make a 3-D projection of your data stack. Do this with the Image>Stacks>Reslice [/] menu item. The input spacing is 1 μm. Set the output spacing to be 0.5-1.0 μm (otherwise the 3-D projection will use too much memory). Remember that your first slice was at the bottom of the cells, and set the start point accordingly. You should end up with a z-x image stack that lets you look at the interior of the cells and their surfaces in cross section. DiI-LDL spot fluorescence power: This will be tedious. For each of the images that represents your best guess as to the cell surface focal plane and the cytoplasmic endosome and lysosome focal planes (this is the second set of images that you took, not the through-focal series) you will measure the total fluorescence power in at least 50 cell surface spots and at least 50 cytoplasmic spots. To measure fluorescence power, which is simply the total fluorescence signal from a spot, you must surround each spot with one of the measuring tools and get the AVERAGE fluorescence signal from it. From that you must subtract the AVERAGE background fluorescence signal (this should be from a representative area free of dii- LDL spots but in the cell. This may be tricky to do, and this value may vary across the cell. You should check it at various points and use a background value that is valid near the dii-ldl spot you are currently analyzing. Once you have subtracted the background average from the spot average fluorescence, you will be left with the net spot fluorescence, which should be due only to the dii-ldl in that spot. Multiplying this value by the total area of the spot (gotten from your measurement when you got the average fluorescence in the spot), you will then have the total fluorescence power emanating from the spot. (You should note that a brighter spot will have more power than a dimmer spot and a bigger spot will have more power than a smaller spot. This is because more dii fluorophores are found in the brighter or larger spots.) These calculations may be easier to do by using a spreadsheet such as Excel. You need to be very systematic when collecting the spot data. You want to sample typical spots and not to resample the same spot. You will need enough data points to make a histogram later on in the lab report. 50 is probably a minimal number to do this. The histogram plot will be described in the lab report description once we have the raw data for all groups. Number of dii-ldl spots per square micron: This image analysis will let you estimate the density of coated pits with LDL-Rs on the cell surface and the density of LDL- 6.10
containing vesicles in the cytoplasm. Open an image that was for an area focused at the cell surface. Set the calibration in μm per pixel for this image (Analyze>Set Scale using the data you have for this objective from Module 3). Using the freehand drawing tool in ImageJ, draw around the area that you think is in focus on a cell surface in the image. Then get the area in μm 2 by using the Analyze>Measure tool in ImageJ. Count the number of dii-ldl spots in this area. Record these data for all cells in the image. There is a data entry form on the Bioimaging web site called LDL spot density ; enter your data there, including the information called for in the form. Each cell will have one set of entries. Repeat this for images of the cells with the focal plane at the cell interior. In this case, it will be more difficult to assess which organelle is in focus. You should make a rule for yourself about what you consider to be in focus and stick to it. Outline the full area of a cell, get its value in μm 2 and count the number of in-focus fluorescent organelles that you find in that area. Enter these data in the database. Repeat this process for all cells and all three cover slips. These accumulated data will be used in the lab report to analyze the movement of dii- LDL from cell surface to cytoplasm. 6.11